Anti-inflammatory Effect of Novel 2-Phenylphthalazin-2-ium Bromides on LPS-induced RAW264.7 Cells and their Mechanism

aims: On this foundation, in this work, we further investigate the therapeutic effects of 2-phenylphthalazin-2-ium bromides on the macrophage-mediated LPS-mediated inflammatory response as well as underlining its molecular mechanisms. background: Quaternary benzo[c]phenanthridine alkaloids (QBAs) have extensively been studied in functions for growth promotion effect and the immune function of the body enhancement, so they have been developed as animal feed additive and attracted the interests of pharmacologists for their very low toxicity to mammals. Meanwhile, quaternary benzo[c]phenanthridine alkaloids such as sanguinarine (SA) and chelerythrine (CH) had been proven to have excellent anti-inflammatory activity. Nevertheless, as potential pant-based antitumor drugs, most of QBAs didn't satisfy our demand for their low content in nature. Thus, it is necessary to develop an effective method to synthesize novel chemical entities with similar structure. Our previous study demonstrated that a novel antifungal 2-phenylphthalazin-2-ium scaffold as a simple analogue was designed and characterized, indicating that they exhibited excellent activity. Most of the compounds showed excellent inhibition activity against almost all eight phytopathogenic fungi, far superior to sanguinarine and chelerythrine. objective: Inspired by quaternary benzo[C]phenanthridine alkaloids, novel 2-phenylphthalazin-2-ium bromides were previously designed and synthesized. The anti-inflammatory effect of 2-phenylphthalazin-2-ium bromides were evaluated based on inflammatory cytokines, and their possible mechanism was explored through NF-κB, TLR4 and MAPK signaling pathways. method: The safe dose range of 2-phenylphthalazin-2-ium bromides was tested by using MTT assay. Griess assay was used to determine the changes of nitric oxide (NO) in the cell culture supernatant. qRT-PCR was used to detect the mRNA levels of inflammatory cytokines, such as IL-6, IL-1ß, IL-10, TNF-α, TLR4 and iNOS. The secretion level of TNF-α and IL-1ß was detected by ELISA. Western blot was used to detect the protein expression of IL-6, IL-10, TLR4, iNOS, NF-κB, p-P38/P38, p-ERK/ERK and p-JNK/JNK. result: 2-(3,5-Dichlorophenyl)phthalazin-2-ium Bromide (2) with a concentration below 1 μg/mL showed no significant effect on the growth inhibition of RAW264.7 cells, so the concentrations of compound 2 used for experiments were set to 0, 0.25, 0.5 and 1 μg/mL. Compared with the blank control group, the model group showed increased release of NO, transcription levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4 and iNOS (P<0.05), and ratios of p-P38/P38, p-ERK/ERK, p-JNK/JNK (P<0.05). Compared with model group, sample groups displayed decreased NO release and reduced transcriptional levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4, iNOS and reducing protein expression ratios of IL-6, IL-1ß, IL-10, TNF-α, NF-κB, TLR4, iNOS, p-P38/P38, p-ERK/ERK and p-JNK/JNK (P<0.05). conclusion: This study showed that 2-phenylphthalazin-2-ium bromides partially protected macrophage from LPS-induced inflammatory response via suppressing the TLR4-NF-κB/MAPK and reducing NO production. other: No